ES2774422T3 - Antibodies to IL-21 - Google Patents

Abstract

An antibody that binds to human IL-21 comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the LCVR comprises SEQ ID NO: 7 in CDRL1, SEQ ID NO : 8 in CDRL2 and SEQ ID NO: 9 in CDRL3 and in which the HCVR comprises SEQ ID NO: 10 in CDRH1, SEQ ID NO: 11 in CDRH2 and SEQ ID NO: 12 in CDRH3, in which the heavy chain comprises an HCVR having SEQ ID NO: 1 and wherein the light chain comprises an LCVR having SEQ.ID.NO: 2.

Description

DESCRIPTION

Antibodies to IL-21

The present invention relates to genetically engineered humanized antibodies that have a high binding affinity and neutralize human IL-21, methods of using the antibodies to treat conditions in which antagonism or neutralization of the effects of IL- is guaranteed. 21, such as autoimmune conditions, compositions and methods for recombinantly producing the antibodies, and pharmaceutical compositions comprising the antibodies.

WO2010 / 055366 discloses human antibodies derived from transgenic mice that are said to bind and neutralize human IL-21. One particular antibody one derived from clone 362.78.

Maurer, M. et al. (Generation and characterization of human anti-human IL-21 neutralizing monoclonal antibodies, 4 MAbs 69 (2012)) describe the generation and initial characterization of a panel of human anti-human IL-21 monoclonal antibodies derived from transgenic mice with human IgG, including the antibody derived from clone 362.78. Clinical studies of an anti-IL-21 antibody described in WO2010 / 055366, US 20130323259A1 and in Maurer et al., Namely "mAb 362.78", which is also known as NN8828 and NNC114-0006 or NNC -00-0006, in patients with systemic lupus erythematosus (SLE) and Crohn's disease.

Document WO2012 / 098113 states that a crystal structure of human IL-21 in complex with a Fab fragment of monoclonal antibody 362.78 (n Nc 0114-0005), the antibody of which had previously been mentioned in document WO2010 / 055366, had been formed and analyzed using X-ray procedures

WO2012 / 164021 discloses human antibodies including one that is derived from clone 366,28.

The present invention disclosed herein seeks to provide alternatives to the anti-IL-21 antibodies described above. The antibodies are potentially well suited for the treatment of autoimmune conditions or diseases such as primary Sjogren's syndrome (SSE), Sjogren's syndrome (SS) and systemic lupus erythematosus (SLE), Graves disease, type 1 diabetes, and others, for which few treatments are available and there is a worldwide medical need. There is a great need for more effective treatments that have better safety profiles than current standards of patient care.

In particular, the present invention provides antibodies having as the heavy and light chain variable domains the engineered and humanized heavy chain variable domain and the light chain variable domain of the antibody referred to herein as Ab327.

The present invention provides antibodies that have one or more of the following properties: 1) bind with high affinity to human IL-21 and to cynomolgous monkey IL-21 (Kd = 0.8 ± 0.5 x 10'12 M and 0.3 ± 0.1 x 10.12 M, respectively, at 37 ° C by solution equilibrium binding KinExA); 2) bind with modest affinity to mouse and rat IL-21 (Kd = 2.4 ± 1.3 x 10.7 and 2.3 ± 0.2 x 10.7 M; respectively, at 37 ° C by the solution equilibrium binding KinExA); 3) does not bind substantially to any other member of the human and common chain family (IL-2, IL-4, IL-7, IL-9, and IL-15); 4) neutralize human monkey and cynomolgous IL-21 activity in a particular pan-STAT-IM9-Luciferase reporter assay with an IC50 approximately 6 times lower than the positive control, a hIL-21R-Fc construct (~ 46, 7 pM and -41pM vs. ~ 271 pM, respectively); 5) neutralize the human IL-21 induced proliferation of primary human B cells in vitro with an IC50 of approximately 1.15 nM; 6) neutralizing human IL-21-induced plasma cell differentiation from primary human B cells in vitro; 7) effectively block the rapid and transient expansion of various cell types in the spleen (including B and T cell subpopulations) in mice injected with human IL-21; and / or 8) are sufficiently stable for pharmaceutical manufacture, storage and therapeutic use.

According to a first aspect of the present invention, there are provided antibodies that bind to human IL-21 comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR) wherein the LCVR comprises SEQ ID NO: 7 in CDRL1, SEQ ID NO: 8 in CDRL2 and SEQ ID NO: 9 in CDRL3 and wherein the HCVR comprises SEQ ID NO: 10 in CDRH1, SEQ ID NO: 11 in CDRH2 and SEQ ID NO: 12 in CDRH3, wherein the heavy chain comprises an HCVR having SEQ ID NO: 1 and wherein the light chain comprises an LCVR having SEQ ID NO: 2.

The present invention further provides human IL-21 binding antibodies comprising two antibody heavy chains and two antibody light chains, wherein each heavy chain comprises a heavy chain variable domain, the amino acid sequence of which is the sequence of SEQ ID NO: 1, and wherein each light chain comprises a light chain variable domain, the amino acid sequence of which is the sequence of SEQ ID NO: 2.

Preferably, the antibody of the present invention consists of two antibody heavy chains and two antibody light chains are provided, in which each heavy chain comprises a heavy chain variable domain, the amino acid sequence of which is the sequence of SEQ ID NO: 1, and wherein each light chain comprises a light chain variable domain, the amino acid sequence of which is the sequence of SEQ ID NO: 2.

A particular embodiment is the Ab327 antibody, which is a manipulated and humanized antibody to human IL-21, whose amino acid sequence of each heavy chain is the sequence of SEQ ID NO: 3 and the amino acid sequence of each light chain of the what is the sequence of SEQ ID NO: 4.

The present invention also provides pharmaceutical compositions comprising the antibodies of the invention and a pharmaceutically acceptable excipient.

The present invention also includes DNA molecules encoding the heavy and light chains of the antibodies of the present invention to express the antibodies of the present invention. In particular, there is provided a DNA molecule comprising a polynucleotide encoding the heavy chain of the antibody whose amino acid sequence is the sequence of SEQ ID NO: 3 and comprising a polynucleotide encoding the light chain of the antibody whose amino acid sequence is the sequence of SEQ ID NO: 4. The present invention further provides a DNA molecule comprising a polynucleotide whose sequence is the sequence of SEQ ID NO: 5 and comprising a polynucleotide whose sequence is the sequence of SEQ ID NO: 6.

The present invention includes mammalian cells for expressing the antibodies of the invention by recombinant means. In particular, according to another aspect of the present invention, there is provided a mammalian cell transformed with a DNA molecule of the invention described above.

Another aspect of the present invention includes a method for producing an antibody, comprising two antibody heavy chains and two immunoglobulin light chains, wherein the amino acid sequence of each of the two heavy chains is the sequence of SEQ ID NO. : 3 and the amino acid sequence of each of the two light chains is the sequence of SEQ ID NO: 4, and which method comprises: a) culturing a mammalian cell of the invention, as described above, under conditions such that the antibody is expressed and b) recovering the expressed antibody. The present invention includes an antibody obtainable by the method of the invention as described immediately above.

In accordance with yet another aspect of the present invention, an antibody of the present invention is provided for use in therapy.

Preferably, the antibody of the present invention is for use in treating an autoimmune condition.

More preferably, the antibody of the present invention is for use in treating primary Sjogren's syndrome, Sjogren's syndrome, systemic lupus erythematosus, Grave's disease, or type 1 diabetes.

The antibody of the present invention is even more preferably for use in the treatment of Primary Sjogren's Syndrome or Sjogren's Syndrome and is more preferably for use in the treatment of systemic lupus.

The present invention includes methods for using the antibodies, particularly to treat autoimmune conditions (AI), especially primary Sjogren's syndrome (SSE), Sjogren's syndrome (SS), systemic lupus erythematosus, Grave's disease, or type diabetes. 1, methods for preparing the antibodies, polynucleotides encoding the antibodies, vectors comprising the nucleotides for transforming the host cells and for expressing the antibodies, host cells for expressing the antibodies, antibodies prepared by a method of recombinant expression in mammalian host systems and pharmaceutical compositions of the antibodies comprising the antibody and a pharmaceutically acceptable carrier.

Particular intended uses for the antibodies are in the treatment of AI conditions, particularly AI conditions such as primary Sjogren's syndrome (SSE), Sjogren's syndrome (SS), systemic lupus erythematosus, Grave's disease, or type 1 diabetes. The present invention includes antibodies for use in therapy, in the treatment of autoimmune conditions, in the treatment of primary Sjogren's syndrome (SSE), Sjogren's syndrome (SS), systemic lupus erythematosus, Grave's disease or type 1 diabetes, and particularly in the treatment of primary Sjogren's syndrome (pSS), Sjogren's syndrome (SS) or systemic lupus erythematosus. The present invention includes the use of an antibody of the invention for the manufacture of a medicament for use in treating an autoimmune condition; for use in the treatment of Primary Sjogren's Syndrome (pSS), Sjogren's Syndrome (SS), systemic lupus erythematosus, Grave's disease, or type 1 diabetes; to treat primary Sjogren's syndrome (pSS), Sjogren's syndrome (SS); or to treat systemic lupus erythematosus. In particular, the present invention includes the use of an antibody of the present invention in the manufacture of a medicament for the treatment of an autoimmune condition; Primary Sjogren's syndrome (pSS), Sjogren's syndrome (pSS), systemic lupus erythematosus, Grave's disease, or type 1 diabetes; Primary Sjogren's syndrome (pSS), Sjogren's syndrome (SS); or systemic lupus erythematosus.

Brief description of the figures

Figure 1. The binding of Ab327 to human IL-21 and other ligands that bind to the human common gamma chain receptor is shown by ELISA.

Figure 2. Ab327 is shown to neutralize human IL-21-induced proliferation of primary human B cells in vitro.

Figure 3. Average percent change in body weight from baseline in severely immunocompromised NSG mice (IL-2R and NOD-scid null) grafted with human peripheral blood mononuclear cells (PBMC) treated with antibody to IL-21 or control antibody to isotype for 42 days. The treatments occurred at the points indicated by arrows. Symbols: - ■ - Ab327 (10 mg / kg); - ▼ - Ab327 (1 mg / kg); - □ -; Isotype control (10 mg / kg); - • - without graft.

Figure 4. Average percent change in body weight from baseline in severely immunocompromised NSG mice (IL-2R and NOD-scid null) grafted with human peripheral blood mononuclear cells (PBMC) treated with antibody to IL-21 or control antibody to isotype. The treatments occurred at the points indicated by arrows. Symbols: - ■ -Ab327 (10 mg / kg); - □ -; Isotype control (10 mg / kg); - • - without graft.

Figures 5 and 6. Treatment with anti-mouse IL-21 antibody shows that it alleviates lymphocytic infiltration in the salivary glands of NOD mice by mRNA analysis (Figure 5) and histological analysis (Figure 6, lymphocytes are summarized).

Figures 7A and 7B. Surrogate antibody 728 has been shown to prevent the development of autoimmune diabetes in NOD mice. Treatment begins at 7 weeks of age (A) as prevention or at 13 weeks of age (B) during the prediabetic phase. The incidence of diabetes is calculated per group as two consecutive readings of more than 250 mg / dl and is shown as the percentage of diabetic mice per group (A: mIgG1 n = 12; Ab728 n = 12 and B: mIgG1 n = 8; Ab728 n = 11). The incidence of diabetes is graded as survival curve data and is different by Logrank's test (A: p = 0.0007 and B: p = 0.002).

Use of Antibodies of the Invention

Interleukin (IL) -21 is produced by various subsets of T cells and binds to a composite receptor consisting of a specific receptor, termed the IL-21 receptor (IL-21R) and the common y-chain subunit. Human IL-21 is produced in vivo from a 162 amino acid precursor molecule. Mature human IL-21 consists of residues 30-162 (133 amino acids) of the precursor protein. Typical of class I cytokines, IL-21 has a bundle structure of four helices arranged in a top-up-down-bottom topology. Using the 162 amino acid precursor protein numbering, the helices are believed to consist of the following residues: Helix A: 41-56, Helix B: 69-84, Helix C: 92-105 and Helix D: 135-148 This Structure is closely related to that of other members of the type 1 cytokine family, especially IL-2, IL-4, and IL-15.

Upon binding of IL-21 to the receptor complex and subsequent activation of the receptor, signaling occurs through the Jak-STAT signaling pathway. The IL-21R chain binds IL-21 with high affinity and provides most of the binding energy. However, interaction with the chain and common is necessary for signaling. The interaction between IL-21 and IL-21R is mediated by residues present in the A and C helices and by a small part of the CD loop that immediately follows the IL-21 C helix (O. Hamming, et al., Crystal Structure of Interleukin-21 Receptor (IL-21R) Bound to IL-21 Reveals That Sugar Chain Interacting with WSXWS Motif Is Integral Part of IL-21R, 287 J. Biol. Chem. 9454-9460 (2012)).

IL-21 is a class I cytokine that has pleiotropic effects on innate and adaptive immune responses, such as stimulation of lymphocyte proliferation, promotion of CD8 + T cell and NK cell cytotoxicity, and differentiation of B cells into plasma cells. IL-21 is secreted by activated CD4 + T cells, in particular Th17 and T follicular helper cells, as well as natural killer cells. It plays an important role in promoting the development of Th17 and T follicular helper cells through a forward mechanism. In addition, IL-21 cooperates with other cytokines to increase the cytotoxicity of CD8 + T cells and promotes the proliferation of CD8 + cells in the presence of antigens. IL-21 also influences the production of antibodies by B cells. IL-21 has a number of actions, including increasing T cell proliferation, driving B cell differentiation into memory cells and terminally differentiated plasma cells, and increasing the activity of natural killer cells. In certain human conditions and diseases it may be desirable to block IL-21 activity. In particular, an antibody that blocks the binding of IL-21 to its receptor would be desirable in the treatment of such conditions and diseases.

Given their properties, the antibodies of the present invention are potentially very suitable for the treatment of autoimmune conditions or diseases in which the interactions T cell - B cell, Th17 cells, follicular helper T cells, plasma cells and activated CD8 and NK cells play a predominant pathogenetic role Diseases that easily fit into this category are Primary Sjogren's Syndrome (SSE), Sjogren's Syndrome (SS), and Systemic Lupus Erythematosus (SLE), for which few treatments are available and there is a large medical necessity around the world. Other potential indications include Grave's disease and type 1 diabetes.

Sjogren's Syndrome is a slowly progressive systemic autoimmune disease, seen in 0.5-1.0% of the population, predominantly affecting middle-aged women, although it can occur at any age and in both men and women. in women. Primary Sjogren's syndrome (pSS) and Sjogren's syndrome (SS) are characterized by chronic inflammation of the exocrine glands, particularly the salivary and lacrimal glands. The main features of pSS are oral and ocular dryness ("Sicca syndrome") and the histological hallmark is Focal lymphocytic infiltration of the exocrine glands, which can be determined by a labial minor salivary gland biopsy. Sicca characteristics affect the quality of life and cause local complications in the involved mucosa. Severe dry mouth is an unpleasant and disabling condition. However, extraglandular manifestations occur in many patients and can affect almost any organ. There are currently no disease modifying agents approved to treat PFS and SS. Medications address symptoms and provide supportive therapy. There is a need for more effective therapies with better safety profiles for patients with PFS and SS. Pharmacological intervention with the antibodies of the present invention can affect various aspects of the dysregulated immune system that appear to be causally related to the pathogenesis of SSP and SS, and therefore could provide significant clinical benefit for patients. This therapy can also reduce the need for chronic (non-specific) immunosuppressive agents, providing an improvement in quality of life for patients with PFS and SS.

Treatment and administration

The terms "treatment", "treating" or "treating" and the like include restricting, slowing, stopping or reversing the progression or severity of an existing symptom, condition, disease or disorder in a patient. The term "patient" refers to a human. The term "effective amount" refers to the amount or dose of an antibody of the invention that, after single or multiple dose administration to the patient, provides the desired effect in the patient. An effective amount can be readily determined by the diagnostic assistant physician or health care professional, one of ordinary skill in the art, using known techniques and observing the results. In determining the effective amount for a patient, a number of factors can be considered, including the size, age, and general health of the patient; the specific disease or disorder involved; the severity of the disease, condition, or disorder; the response of the individual patient; the mode of administration; the bioavailability characteristics of the administered preparation; the selected dose regimen; the use of concomitant medication; and other relevant factors.

The antibodies of the invention are intended for parenteral administration to humans to treat autoimmune conditions. The subcutaneous and intravenous routes are preferred. Antibodies can be formulated in aqueous pharmaceutical solutions prior to administration. A loading intravenous dose can be administered by a healthcare professional. In general, administration is expected to be by subcutaneous injection, either self-administered by the patient or by another person, such as a healthcare professional. For subcutaneous injections, an autoinjector or a pre-filled syringe can be used. Doses can be fixed, that is, the same mass of active antibody for each patient, or can be based on body weight (mass). On a body weight (mass) basis, the doses will preferably be in the range of 0.01 to 30 mg of antibody per kilogram of body weight (mass) of the patient. More preferably, the dose will be in the range of 0.1 to 20 mg of antibody per kilogram of body weight (mass) of the patient. The dosing frequency is expected to be weekly or less frequent, depending on the actual pharmacokinetics and pharmacodynamics in humans. The duration of treatment will vary depending on many factors and will be determined by the patient's diagnosis or the treating healthcare provider, based on experience and skill in the art. The frequency and duration of treatment may vary depending on the indication.

Structure of the antibodies of the invention.

The antibodies of the invention have a typical tertiary and quaternary structure for a full-length human IgG antibody. When biosynthesized in a suitable mammalian host cell, the antibodies of the invention will be secreted as molecules consisting of two heavy chains and two light chains, which chains are covalently linked via intrachain and interchain disulfide bonds in the usual manner, with the chains heavy chains attached to each other and a light chain attached to each of the heavy chains. The positions of the intrachain and interchain disulfide bonds are well known.

Each heavy chain contains four domains: N-terminal to C-terminal, heavy chain variable domain, IgG CH1, IgG CH2, and IgG CH3 domains. A hinge region between CH1 and CH2 contains an interchain disulfide bond or bonds that join the two heavy chains. Each light chain contains two domains: from terminal N to terminal C, light chain variable domain and a light chain constant domain (CL) domain. It is preferred that the heavy constant domains are human IgG4 or variants thereof. It is preferred that the light constant domain is human kappa. Together, the variable regions are responsible for the functional properties of binding to human IL-21 and neutralizing the activity of human IL-21. The amino acid sequences of the variable regions of the antibodies of the invention are provided in SEQ ID NO: 1 and SEQ ID NO: 2. The constant domains of an antibody of the invention, Ab327, are provided within SEQ ID NO: 3 and SEQ ID NO: 4. The N-linked glycosylation site in Asn294 of SEQ ID NO: 3 may be glycosylated.

The amino acid sequence of the heavy chain (HC) of the antibodies consists of the heavy chain variable domain of the present invention, SEQ ID NO: 1, fused at its C-terminus to constant domains of the appropriate human heavy chain (CH1 , CH2 and CH3) or structurally similar variants of the human heavy chain constant domains that may have a well-known mutation or mutations to improve stability or reduce effector functions. Variants of human IgG4 constant domains are preferred having mutations related to stability and / or reduced effector function. SEQ ID NO: 3 provides the amino acid sequence of the Ab327 heavy chain.

The light chain (LC) amino acid sequence consists of the light chain variable domain of the present invention, SEQ ID NO: 2, fused at its C-terminal to an appropriate human light chain (CL) constant domain or a structurally similar variant of it. The constant domain of the human kappa light chain is preferred. SEQ ID NO: 4 provides the amino acid sequence of the Ab327 light chain. To express Ab327, the DNA sequences provided by SEQ ID NO: 5 and SEQ ID NO: 6 can be used for HC and LC, respectively.

CDRs L1, L3 and H2 were assigned according to the Kabat convention and CDRs L2, H1 and H3 were assigned according to the North convention. Kabat EA, et al., Sequences of Proteins of Immunological Interest, Fifth Edition, U.S. Department of Health and Human Services, NIH Publication No. 91-3242 (1991); North B, et al, J Mol Biol 2011 Feb 18; 406 (2): 228-256.

Production of the antibodies of the invention.

The antibodies of the present invention can be biosynthesized, purified, and formulated for administration by well known procedures. An appropriate host cell, such as HEK 293 or CHO, is transiently or stably transfected with an expression system to secrete antibodies using a predetermined HC: LC vector ratio if two vectors are used, or a single vector system encoding both the heavy chain as the light chain. Suitable vectors for the expression and secretion of antibodies from these commonly used host cells are well known.

After expression and secretion of the antibody, the medium is clarified to kill cells and the clarified medium is purified using any of the many techniques in common use. For example, the medium can be applied to a Protein A column that has been equilibrated with a buffer, such as phosphate buffered saline (pH 7.4). The column is washed to remove non-specific binding components. Bound antibody is eluted, for example, by pH gradient (such as 0.1 M sodium phosphate buffer pH 6.8 to 0.1 M sodium citrate buffer pH 2.5). Antibody fractions are detected, as by SDS-PAGE, and then pooled. Additional purification is optional, depending on the intended use. The antibody can be concentrated and / or filtered to sterility using common techniques. Materials other than the antibody, such as the host cell and growth medium components, and the soluble aggregates and multimers of the antibody, can be effectively reduced or removed by common techniques, including size exclusion chromatography, hydrophobic interaction , cation exchange, anion exchange, affinity or in hydroxyapatite. The purity of the antibody after these chromatography steps is typically greater than 95%. The product can be stored at 4 ° C, frozen at -70 ° C or can be lyophilized.

The Ab327 used in the studies described herein was transiently expressed in HEK293 cells after cotransfection of separate heavy chain and light chain expression DNA vectors that incorporated the DNA sequences of SEQ ID NO: 5 and SEQ ID. NO: 6, respectively, or was stably expressed in CHO cells after transfection of a single DNA vector incorporating the DNA sequences of SEQ ID NO: 5 and SEQ ID NO: 6, which encode the heavy chain and the light chain, respectively. The medium harvested from a 5-day-old HEK293 culture or a 14-day CHO bulk culture was rinsed and the resulting crude supernatant was purified by Protein A chromatography. Ab327 was bound to Protein A resin and eluted using buffer. low pH. The eluted antibody was further purified using preparative size exclusion chromatography (SEC), for material produced from transient transfection of HEK293, or using multimodal anion exchange chromatography (Capto adhere® GE Healthcare Life Sciences) as a purification step to the material produced from stable CHO transfection. Final purity of Ab327 was evaluated by s DS-PAGE, SEC-HPLC analytical and LC / MS analysis. Endotoxin levels were shown to be <1 EU / mg using Endosafe-PTS assay. The purified Ab327 was stored in PBS (phosphate buffered saline), pH 7.2 at 4 ° C.

Pharmaceutical Compositions of the Antibodies of the Present Invention

The purified antibody can be formulated into pharmaceutical compositions according to well known procedures for formulating proteins and antibodies for parenteral administration, particularly for subcutaneous or intravenous administration. The antibody can be lyophilized, along with appropriate pharmaceutically acceptable excipients, and then reconstituted with a water-based diluent prior to use. Alternatively, the antibody can be formulated in an aqueous solution and stored for up to 1 to 3 years before use. In any event, the stored form and the injected form of the antibody pharmaceutical compositions will likely contain a pharmaceutically acceptable carrier (s), which are ingredients other than the antibody. Whether an ingredient is pharmaceutically acceptable depends on its effect on safety and effectiveness, or on the safety, purity, and potency of the pharmaceutical composition. If an ingredient is considered to have a sufficiently unfavorable effect on safety or effectiveness (or safety, purity, or potency) to ensure that it is not used in a composition for human administration, then it is not pharmaceutically acceptable for use in a pharmaceutical composition of the antibody.

A solution composition of the antibody will typically be water-based and may contain, in addition to water, excipients such as a buffer system, a preservative if intended for multiple uses, a chelating agent, a tonicity agent to adjust the tonicity of the composition to approximate that of human tissue, and / or a solubilizing or stabilizing agent (s), such as a detergent, a surfactant, or the like. Achieving adequately stable formulations for long-term storage in solution can be challenging. Solubility and chemical and physical stability can be improved using experiment design procedures by varying, for example, pH, the inclusion (or not) of various excipients, and the type and concentration of excipients. A source for general information on drug formulation is Remington: The Science and Practice of Pharmacy. W. Wang, et al., Antibody Structure, Instability, and Formulation, 96 J. Pharm. Sci. 1-26 (2007) which is a useful general source for formulating antibodies.

Various features and preferred embodiments of the present invention will now be described by way of example only and non-limiting testing with reference to the accompanying figures.

Example 1

Generation of antibodies, manipulation and humanization to obtain Ab327

A mouse anti-human IL-21 antibody, 15H12, was isolated after mouse pad immunizations and cloning of anti-IL-21 variable regions. Balb / c mice were immunized using standard immunization procedures with commercially obtained human IL-21. Three to five days after the final non-adjuvant boost, lymph nodes and / or spleen were harvested, and single-celled suspensions were generated. Antigen-specific cells were enriched by standard sorting procedures using biotinylated or fluorophore-labeled IL-21 and co-cultured with feeder cells for two weeks prior to cloning of mouse variable domains. The antibody was identified using an anti-IL-21 capture ELISA and was shown to block and neutralize IL-21 in in vitro assays, with a Kd of approximately 5 pM for human iL-21 and IL-21 from cynomolgus monkey. Fab antibody fragments were captured with a goat anti-human kappa antibody and then screened for the ability to bind biotin-labeled IL-21 which in turn was detected by alkaline phosphatase-labeled neutravidin.

The antibody was further optimized to bind both mouse and human IL-21 to produce the 15M2 mouse antibody. To accomplish this, the c Drs of isolated murine 15H12 VHs and VLs were randomized by mutagenesis and the resulting antibodies were selected for binding to human and mouse IL-21 using an ELISA. The affinity enhancing mutations were combined to produce 15M2, which were then humanized using framework library methodology. For the framework library, twelve human VH framework germline genes (1-24, 1-46, 1-69, 2-5, 3-15, 3-23, 3-53, 3-72, 4-04, 4-39, 5-51 and 6-01) and eight human VL framework genes (A-19, A-26, A-27, B-2, B-3, L-2, L-12 and O-2 ) containing 15M2 CDRs were synthesized and cloned into human heavy and light chain IgG4 expression vectors. After transient transfection with 293 HEK of the 96 combinations of heavy and light chains, the supernatants were analyzed by ELISA for binding to human IL-21 coated directly on a plate and to biotinylated IL-21 in solution after capture of Human IgG from supernatants with an anti-human kappa antibody. Considering the extensibility and activity of ELISA, a humanized antibody with CDRs derived from the 15M2 antibody was chosen, using the human heavy chain framework 1-46 and the human light chain framework O2, for further optimization. Its Kd for human IL-21 was approximately 0.5 pM.

Analysis of the humanized antibody identified a light chain CDR deamidation site (Asn92) and a heavy chain CDR isomerization site (Asp55). To eliminate these critical chemical degradation points, an engineered antibody containing mutations of the heavy chain Asp55Glu and the light chain Asn92His was generated. These mutations caused some loss of affinity, at approximately 2 pM Kd for human and cynomolgus IL-21 monkeys. The loss of affinity was determined to be attributed to the Asp55Glu mutation. As a result, the Asp55 residue remained on the heavy chain along with the Asn92His mutation on the light chain. Rational engineering principles guided further optimization of affinity by engineering the nearby Ser56 neighbor residue in the heavy chain. From this, an engineered antibody was selected that contained a Ser56Val mutation that simultaneously reduced Asp55 isomerization and improved affinity. The final antibody has an affinity of approximately 0.5 pM for both human and cynomolgus monkey IL-21. The constant region was selected to be human IgG4, with point mutations to prevent the formation of half antibody (S225P) and to reduce effector functions (F231A / L232A). Epivax in silico assay showed no discernible immunogenicity critical points. The sequence information for the final engineered humanized antibody ("Ab327") is shown in Table 1 below.

Table 1

(continuation)

Solubility and stability characteristics of Ab327

The physicochemical properties of Ab327 related to solubility and stability were evaluated in formulations of 10 mM citrate, pH 6 (C6) and 10 mM citrate, pH 6 150 mM NaCl (C6N). For formulation C6, the solubility was> 122.9 mg / ml and the solubility in formulation C6N was> 130.9 mg / ml. The viscosity of Ab327 at 100 mg / ml in 10 mM citrate, 150 mM NaCl, 0.02% Tween 80, pH6 (C6NT) was determined to be 5.8 cP.

After 4 weeks at 25 ° C in 10 mM citrate, pH 6 buffer, changes in chemical and physical heterogeneity were low for Ab327, with less than 5% change in the area of the main peak observed by exchange chromatography. cationic analytical (CEX). Under the same conditions, aggregate growth of <0.5% high molecular weight (HMW) was observed by analytical exclusion chromatography (SEC). Citrate samples with pH 6 stability showed no loss of bioactivity in a cell-based assay after 4 weeks at 40 ° C.

The antibody was analyzed after 4 weeks at 4 ° C, 25 ° C, and 40 ° C in C6 by peptide mapping by LC-MS for the characterization of critical points of chemical degradation of the CDR. LC-MS analysis showed no significant isomerization of the heavy chain CDR residue Asp55 at 40 ° C relative to the control sample at 4 ° C. Other common CDR degradations were identified that grew by more than 0.5% after 4 weeks at 25 ° C relative to the 4 ° C control sample and collectively in the 6 CDRs totaled less than 5%.

The physical stability of Ab327 was evaluated with a 1 mg / ml fast freeze-thaw study and a 50 mg / ml slow-thaw study in C6. During both studies, the presence of Tween 80 reduced the formation of 10 micron size particles. In the high concentration study, the presence of 150 mM NaCl salt reduced the formation of HMW aggregates. For the low concentration study, the HMW addition was 0.7% in the presence of salt and Tween 80. In conclusion, Ab327 appeared to have good solution properties, including viscosity, solubility, and stability, to proceed with human studies.

Functional properties of Ab327

Binding of Ab327 to human IL-21 and other members of the human common gamma chain receptor family by ELISA

The aim of this study was to determine the binding specificity of Ab327 to human IL-21 compared to the other members of the common gamma chain receptor (yc) cytokines. The cytokine receptor yc chain family consists of six members, IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. All members of this family signal through receptor complexes that contain the yc subunit. The specificity of Ab327 for binding to other members of the human yc chain cytokine family was determined using an ELISA. Briefly, Ab327 was captured on an ELISA plate coated with goat anti-human IgG kappa. Biotin-labeled cytokines titrated from 100 nM-780 pM were then added to the plate for 1 hour at 37 ° C and any bound cytokines were detected using alkaline phosphatase-labeled Neutravidin. Commercially available mouse monoclonal antibody to IL-2 and goat polyclonal antibodies to IL-4, IL-7, IL-9, and IL-15 captured with anti-donkey anti-goat IgG gave positive signals after binding of biotinylated cytokines and Neutravidin detection. However, no detectable signal was observed for Ab327, except with human IL-21 (see Table 2 below, which shows the binding of Ab327 and other ligands that bind to human common gamma chain receptor by ELISA as shown as optical density (OD) measurement at 560 nm (see also Figure 1). These results indicate that Ab327 is specific for IL-21 and does not bind to other cytokines of the human y-chain receptor family.

Table 2

Ab327 binding affinity for human, Cynomolgus monkey, mouse, rat, and rabbit IL-21

Ab327 is an engineered humanized monoclonal IgG4 antibody that binds to and neutralizes human IL-21. The purpose of this study was to determine the binding affinity of Ab327 for human, cynomolgus monkey, mouse, rat and rabbit IL-21 (sequences are given below). The apparent binding affinity (Kd) of Ab327 to these various IL-21 species was determined using a KinExA 3000 instrument at 37 ° C. Equilibrium binding experiments in KinExA solution were performed using a protocol of fixed antibody concentration and 2-fold serial dilutions of IL-21. The samples were equilibrated at 37 ° C for 6-36 hours before analysis. Free antibody in balanced samples was detected using a Dylight 649-conjugated anti-human IgG Fc polyclonal antibody. The resulting percent free antibody versus antigen concentration data was fitted to a "standard affinity" binding model using the software. KinExA Pro, and the best binding affinity (Kd) value was determined.

The average Kd from three independent experiments along with the standard deviation (human, cynomolgus monkey, mouse and rat) or from a single determination (rabbit IL-21) is summarized in Table 3 below (apparent binding affinity at equilibrium in solution (Kd) of Ab327).

Table 3

(continuation)

Ab327 bound to human and cynomolgus monkey IL-21 with a mean affinity (n = 3) of 0.8 ± 0.5x10.12 M and 0.3 ± 0.1x10.12 M, respectively, at 37 ° C. Ab327 bound mouse, rat, and rabbit IL-21 with an affinity of 2.4 ± 1.3x10-7 M, 2.3 ± 0.2x10.7 M, and> 2x10-7 M, respectively. Binding to rabbit IL-21 was estimated based on a binding signal that was less than that observed for equivalent samples of mouse and rat IL-21. Based on these results, Ab327 has approximately picomolar level affinity for human and cynomolgus monkey IL-21, but relatively weak affinity for mouse, rat, and rabbit IL-21.

Ab327 inhibited human and Cynomolgus monkey IL-21 in an in vitro pan-STAT-Luciferase IM9 reporter assay.

IL-21 activates JAK family protein tyrosine kinases that mediate IL-21-dependent activation of the Signal Transducer and Activator of Transcription (STAT). The ability of IL-21 to activate the STAT pathway was evaluated using IM9 cells. IM9 cells, which are an EBV-transformed B lymphoblastoid cell line derived from the blood of a multiple myeloma patient and naturally expressing the IL-21 receptor (IL-21R) and its coreceptor (yc), were stably transfected with a pan-STAT-luciferase reporter construct. Using an IM9-panSTAT-luciferase reporter assay, the purpose of this experiment was to determine whether Ab327 could inhibit IL-21-dependent activation of STAT.

IM9-panSTAT-luciferase cells (IM9 1B10 / 3G2 cell subclone with pan-STAT luciferase reporter) were routinely cultured in medium (RPMI1640, 10% FBS, 1X pen / streptococcus, 100 pg / ml zeocin for selection of pan-STAT-luciferase reporter) in flasks. For the assay, cells were seeded at 50,000 cells / 50 µl / well in TC-treated plates and incubated overnight at 37 ° C. The cells were then treated with Ab327 in the presence of recombinant IL-21 proteins from different species. A dose range of Ab327 from 0 to 6670 pM was evaluated (final concentration was based on MW of Ab327 = 150 kDa). Recombinant IL-21 from different species was added to each well at a final concentration of 66.67 pM (based on MW = 15 kDa). A human IL-21R: Fc chimera (R&D Systems, cat # 991-R2) was used as a positive control and human IgG4 was used as a negative control. A dose range for the positive and negative control was evaluated from 0 to 47520 pM. The tests were carried out in triplicate. The 96-well plates were placed in a tissue culture incubator (37 ° C, 95% relative humidity, 5% CO2) for 4 hours. 100 µl / well of Luciferase One-Glo solution was added to stop the assay. A luminometer (Perkin Elmer Victor3) was used to read the plates.

Results are expressed as IC50 (half the maximum inhibitory concentration) and are calculated using a 4-parameter sigmoidal fit of the data (Sigma plot). The average IC50 of three independent experiments and the standard deviations are presented in Table 4 below.

Table 4

Within the range tested, Ab327 completely inhibited human and cynomolgus monkey IL-21-induced STAT activity in a dose-dependent manner. The inhibition by Ab327 was greater than that observed with the positive control (hIL-21R: Fc), with Ab327 with an IC50 of 46.7 ± 2.4 versus 271 ± 15.6 for the positive control. The isotype control antibody (hIgG4) did not inhibit pan-STAT activity (data not shown). In conclusion, Ab327 neutralized indeed human and cynomolgus monkey IL-21 activity in vitro, but did not neutralize mouse, rat or rabbit IL-21 (sequences given above) under these conditions (NND = neutralization not detected).

Ab327 neutralized the human IL-21-induced proliferation of primary human B cells in vitro.

The main function of B cells is to produce antibodies that neutralize and kill pathogens. Antibody-producing B cells are generated from naive B cells during germinal center (GC) reactions. GCs are established when B cells encounter specific antigens and receive instructional signals from helper follicular T cells for growth, survival, selection, and differentiation. Among those signals, B cells are stimulated by CD40 and numerous cytokines, with IL-21 being a key factor in promoting proliferation, isotype switching, plasma cell differentiation, and antibody secretion. The goal was to determine whether Ab327 could inhibit IL-21-induced proliferation of primary human B cells.

Buffy coats were obtained from five healthy donors from the Indiana Blood Center. PBMCs were isolated from buffy coats by Ficoll-Paque gradient separation and CD19 + B cells were positively selected with anti-CD 19 magnetic beads (Miltenyi Biotec). The purity of the recovered population was typically> 90%. To assess the proliferative responses of cultured cells, purified CD19 + cells were grown at 0.5 million cells / ml (0.1 million cells / well) in 96-well flat-bottom culture plates with appropriate stimulators (RPMI-1640 / 10% FBS containing 1 mM sodium pyruvate, non-essential amino acids, 10 mM HEPES pH 7.0, 100 U / ml penicillin and 100 pg / ml streptomycin) for 5 days at 37 ° C and 5% CO2. Isolated B cells were incubated with a combination of 3.33 nM human IL-21 (based on MW = 15k Da) and 2 pg / ml anti-human CD40 (BD Pharmingen). A dose range of Ab327 (0.1 nM to 26.7 nM), human IL-21R chimera: Fc (0.1 nM to 213.3 nM, R&D Systems) or human IgG4 (0, 1 nM to 26.7 nM). All stimuli and treatments were added at the beginning of the culture. After 5 days of culture, [methyl-3H] thymidine uptake was measured using a liquid scintillation counter.

The results are expressed as a percentage of maximum proliferation, with stimulation mediated by IL-21 in the absence of antibody being 100%. The concentration where 50% of the IL-21-induced response (IC50) was inhibited by Ab327 was calculated using a 4-parameter sigmoidal fit of the data. Ab327 inhibited IL-21-induced proliferation of primary human B cells in a dose-dependent manner. This inhibition was much greater than that observed with the positive control, a human IL-21R-Fc construct, which, at the highest concentration used (26.7 nM), was not able to completely inhibit the proliferation induced by IL-21. (See Figure 2) The IC50 calculated for Ab327 was 1.15 ± 0.25 nM (average of 5 independent experiments ± SD). The negative control antibody (hIgG4 isotype control) did not inhibit IL-21-induced proliferation of primary B cells. Table 5 shows that Ab327 is capable of neutralizing human iL-21-induced proliferation of primary human B cells in vitro. The numbers show the percent proliferation of human B cells ± SDEV. In conclusion, Ab327 inhibited IL-21-induced proliferation of primary human B cells in vitro.

Table 5

(continuation)

Ab327 neutralized human IL-21-induced plasma cell differentiation from primary human B cells in vitro.

The differentiation of B cells into plasmablasts is regulated by the integration of signals provided by antigen and T cells (CD40-CD40L interaction and cytokine production). One of the important cytokines for the differentiation of human B cells is IL-21, which induces the generation of plasma cells and the secretion of antibodies from activated memory and naive B cells. IL-21 has been shown to induce the expression of CD25 (IL-2R) in activated B cells, sensitizing those cells to the differentiation promoting effects of IL-2, thus allowing cooperative interaction between IL-2 and IL -21 to amplify plasmablast generation and antibody secretion. The objective was to determine whether Ab327 could inhibit the IL-21-induced differentiation of primary human B cells into plasma cells in vitro.

Buffy coats were obtained as described above. Purified B cells were grown at 0.75 million cells / ml (0.15 million cells / well) in 96-well flat-bottom culture plates with appropriate stimulators (RPMI-1640/10% FBS containing pyruvate 1 mM sodium, non-essential amino acids, 10 mM HEPES pH 7.0, 100 U / ml penicillin and 100 pg / ml streptomycin) for 6 days at 37 ° C and 5% CO2. The isolated B cells were incubated with a combination of 3.33 nM human IL-21, 1 pg / ml anti-human CD40 (BD Pharmingen), 100 U / ml human IL-2 (Proleukin, Hanna's Pharmaceutical Supply Co.) and / or 26.7 nM Ab327 or 26.7 nM of a human IgG4 antibody (negative control). Six days after culture, cells were washed with staining buffer (2% FBS / PBS) and incubated with antibodies specific for human CD38, IgD, CD19, CD27 (all from BD Biosciences) for 40 minutes at 4 ° C. . Five color flow cytometric analysis was performed using an FC-500 flow cytometer (Beckman Coulter). Plasma cells were identified as cells expressing high levels of c D38 and low levels of IgD (see Table 6 below).

Given that there is high variability between donors, the results shown below are expressed as an increase in the relative number of plasma cells (CD38 high low IgD cells) induced by the addition of IL-21, where the value for cells plasma derived from each donor in anti-CD40 IL-2 medium is set equal to one (1). Data is shown as "multiplicity increase" for effects on primary human B cells from five healthy donors.

Table 6

Fresh B cells cultured with the combination of anti-CD40 and IL-2 contained few high CD38 / low IgD plasma cells. In contrast, costimulation of purified B cells with anti-CD40 and IL-2 in the presence of IL-21 resulted in substantial differentiation into plasma cells. The negative control antibody (Isotype hIgG4) was unable to inhibit IL-21 activity. Ab327 inhibited IL-21-induced plasma cell differentiation of primary human B cells (n = 5 donors, p = 0.008, Ab327 unpaired t-test versus IgG4 isotype antibody). In conclusion, Ab327 inhibited human IL-21-induced plasma cell differentiation in vitro.

Ab327 neutralized human IL-21 activity in mice.

Injection of IL-21 into mice leads to a rapid and transient expansion of various cell types in the spleen (including B and T cell subpopulations) clearly identified using specific markers. The aim of this experiment was to investigate whether Ab327 could inhibit the biological activity of human IL-21 in mice.

Eight to ten week old female C57B16 mice (n = 5 per group) were injected intraperitoneally (ip) with Ab327 (1 mg / mouse) or isotype control antibody (hIgG4, 1 mg / mouse) on day 1. On days 2 and 3 the mice received ip injection of 50 µg of recombinant human IL-21 per mouse per day or PBS. On day 4, a spleen cell cell suspension was prepared and the total number of cells was determined after lysing the red blood cells. The relative percentage of cells sensitive to IL-21 was determined using the cell surface markers Gr-1 and Sca-1 by flow cytometry. The total number of IL-21 responsive cells per spleen was calculated by multiplying the percentage of IL-21 responsive cells (Gr-llowSca-1 + cells) by the total number of cells in the spleen.

The results given in Table 7 below are shown as the total number of IL-21 sensitive cells (x106) in the spleen of each of the five mice.

Table 7

The injection of human IL-21 caused an increase in cells sensitive to IL-21. The presence of Ab327 reduced the number of these cells (p <0.0001, Ab327 IL-21 unpaired t-test vs. IgG4 IL-21 and IgG4 PBS vs. IgG4 IL-21) in relation to the animals that received a control negative for antibodies. Exposure to Ab327 and negative control antibodies within each group was confirmed by quantitative ELISA. It is concluded that Ab327 effectively neutralized the biological activity of human IL-21 in vivo.

Ab327 demonstrated efficacy in an in vivo model of human T cell activation in NGS mice.

Neutralization of human IL-21 has previously been shown to prevent disease progression in a model of human T-cell activation in which human peripheral blood mononuclear cells (PBMC) are grafted onto severely immunocompromised NSG (NOD-) mice. scid IL-2Ry null; Hippen KL, et al. Blocking IL-21 signaling ameliorates xenogeneic GVHD induced by human lymphocytes. Blood 2012; 119: 619). NSG mice lack T, B, and NK cells, and they also have reduced macrophage and dendritic cell function. Transplantation of human PBMC results in open activation of human T cells and their infiltration into the skin, liver, intestine, lungs, and kidneys of mice. This is accompanied by a wasting syndrome that ultimately leads to death (Hippen, 2012). The advantage of this model is that the disease is driven by human immune cells and human cytokines, allowing in vivo interrogation of antibodies that lack cross-reactivity with other species. The purpose of this study was to demonstrate the in vivo efficacy and disease modifying activity of Ab327 in the human T cell activation model when administered in prevention mode (given starting at the time of graft) or treatment mode (given starting 21 days after graft)

Before engraftment, female NSG mice were divided into groups based on baseline body weight measurements (n = 10 / group). On day 0, mice were injected intravenously with 10 7 human PBMC isolated from a buffy coat acquired from the San Diego blood bank. For the prevention mode (Figure 3), mice were dosed subcutaneously with 1 or 10 mg / kg of Ab327 or 10 mg / kg of hIgG4 isotype control antibody at the time of engraftment and once a week from so. Body weight was measured and general appearance and health were monitored 2-3 times a week. On day 19, blood was obtained by tail cutting and the human CD45 + cell graft was analyzed by flow cytometry. For the treatment mode (Figure 4), animals that received no treatment were reassigned into matched cohort groups based on flow cytometric data and body weight. On day 21 post-grafting, mice were dosed subcutaneously with 10 mg / kg of Ab327 or 10 mg / kg of hIgG4 isotype control antibody, and then weekly thereafter. Four ungrafted mice were included as "untreated controls or ungrafted mice". Body weight change was calculated as a percentage of baseline weight (day (x) weight / day 0 weight * 100). Results are shown as a percentage of body weight change from baseline over time.

For the prevention mode, mice treated with a human isotype control antibody (see Figure 3, open squares) developed a wasting phenotype as early as 20 days after cell transfer. On day 45 post-transfer, the average weight loss in the isotype control group was greater than 10% from baseline and most of the mice were in distress, so the study was terminated. Treatment with Ab327 10 mg / kg / week (Figure 3, closed squares) started on the day of graft (prevention mode) completely abolished the wasting phenotype. The mice continued to gain weight comparable to the ungrafted mice. Their weights were statistically significantly different from the isotype control group (p = 0.000846 and p <0.001 Ab327 versus isotype and non-grafted versus isotype mice respectively, 2-way ANOVA, repeated measures). The 1 mg / kg / week dose of Ab327 (Figure 3, closed triangles) had a partial effect, as it appeared to slow the progression of weight loss. However, the weights of the mice in this group were not statistically different from the isotype control. Table 8 shows the mean ± SDEV percentage of body weight change from baseline in severely immunocompromised NSG mice (NOD-scid IL-2Ry null) grafted with human peripheral blood mononuclear cells (PBMC) treated with antibody to IL-21 or sotype control antibody for 45 days. Treatment started at the same time as the PBMC graft. There is a significant difference between the Ab327 10 mg / kg group and the isotype control group.

Table 8

(continuation)

As indicated above, the administration of human PBMC resulted in a degenerative disease that began approximately 20 days after grafting. To investigate whether the blocking of human IL-21 was able to stop the deterioration of the ongoing disease (treatment mode), the mice were treated with 10 mg / kg / week of Ab327 isotype control or hIgG4 from day 21 after grafting. As shown in Figure 4, mice given the human IgG4 isotype control antibody (open squares) continue to lose weight. On the other hand, treatment with Ab327, started after the onset of the disease (21 days after grafting, Figure 4, closed squares), was effective in attenuating the wasting phenotype. The mean weight in this group was statistically significantly different from the isotype control group (p = 0.042, 2-way ANOVA, repeated measures). The difference in weight loss and disease severity was not due to differences in human cell grafting in the mice. Peripheral human CD45 + cell numbers, as well as human cells in the spleen at the end of the study, were slightly higher in Ab327-treated mice compared to isotype control-treated animals. In summary, Ab327 demonstrated efficacy and disease modifying activity in a xenogeneic model of T cell activation in vivo. Table 9 shows the mean ± SDEV percentage of body weight change from day 21 in severely immunocompromised NSG mice (IL-2R and NOD-scid null) grafted with human peripheral blood mononuclear cells (PBMC) treated with antibody to IL-21. or antibody isotype control. Treatment started on day 21. There is a significant difference between the Ab327 10 mg / kg group and the isotype control group.

Table 9

(continuation)

Pharmacokinetics of Ab327

The pharmacokinetics of Ab327 were characterized after a single intravenous or subcutaneous dose of 3 mg / kg in male cynomolgus monkeys. Serum samples were collected up to 1008 hours after dosing (6 weeks). Concentration time profiles were generated after quantifying the antibody using two ELISA procedures (total human IgG or antigen capture). The total human IgG procedure uses an ELISA format to measure the concentration of anti-IL-21 antibody. Standards, controls and test samples were incubated with AffiniPure F (ab ') 2 Fragment Goat Anti-Human IgG (Ab coating) which had been immobilized on a microtiter plate. After incubation, a mouse anti-human IgG4-HRP (horseradish peroxidase) was added to the wells. After the unbound enzyme was washed away, SureBlue® TMB (tetramethylbenzidine) substrate solution was added to the wells. Color development was stopped by adding an acid solution and the optical density was measured at 450 nm with the wavelength correction set at 650 nm.

The antigen capture procedure uses an ELISA format to measure the anti-IL-21 antibody concentration. Standards, controls, and test samples were incubated with human IL-21-biotin that had been immobilized on a streptavidin-coated microtiter plate. After incubation, a mouse anti-human IgG4-HRP (horseradish peroxidase) was added to the wells. After the unbound enzyme was washed away, SureBlue® TMB (tetramethylbenzidine) substrate solution was added to the wells. Color development was stopped by adding an acid solution and the optical density was measured at 450 nm with the wavelength correction set at 650 nm. The assay range was 5-500 ng / ml.

The pharmacokinetic results (means) are provided below in Table 10 and Table 11. The number of animals in each group was 2.

Table 10

Abbreviations = t1 / 2 - half-life, AUC0-t - area under the curve from 0 to the last measurable concentration, AUCO-inf - area under the curve from 0 to infinity, extrapolated AUC% - Percentage of AUCO-inf due to extrapolation from last measurable concentration to infinity, CLss: estimate of total body elimination, Vss: estimate of volume of distribution at steady state. * Terminal half-lives were calculated between 72,168 hours.

Table 11

Abbreviations = t1 / 2 - half-life, Tmax - time at maximum concentration, Cmax - maximum concentration, AUC0-t -area under the curve from 0 to the last measurable concentration, AUCO-inf - area under the curve from 0 to infinity, Extrapolated AUC% - percentage of AUC0-INF due to extrapolation from last measurable concentration to infinity, CLss / F - elimination / bioavailability, F% - bioavailability using an average of 3 mg / kg iv dose as reference = (AUC0-inf s.c./AUC0-infi.v.)/(Dosis-iv/Dosis-s.c.)*100. * Terminal half-lives were calculated between 96-336 hours.

After a single intravenous or subcutaneous administration of Ab327 to male cynomolgus monkeys, concentration-time profiles suggested the formation of anti-drug antibodies (ADA) and ADA was confirmed in 4/4 monkeys. The average terminal half-life was 105-249 hours and was calculated from the slope between 72-168 hours to avoid a significant impact of ADA. The mean clearance was 0.43-0.48 ml / hr / kg, which is just outside a typical monoclonal antibody clearance range of 0.2-0.4 ml / hr / kg.

After subcutaneous administration, the bioavailability was 72-74%, which is in the typical range for a monoclonal antibody (50-100%). Despite ADA formation, the pharmacokinetics of Ab327 in monkeys were relatively similar to that expected for a monoclonal antibody that binds to a soluble ligand with slightly higher than normal clearance.

Based on these studies, it is concluded that Ab327 will have human pharmacokinetics within the range expected for a humanized IgG4 antibody. The projected human clearance is 0.3 ml / hr / kg (0.02 L / hr in a 70 kg human) based on the allometric scale of clearance in monkeys and the bioavailability is projected to be 50-75 % in humans.

Treatment with anti-mouse IL-21 antibody alleviated lymphocytic infiltration in the salivary glands of NOD mice.

Since Ab327 does not neutralize rodent IL-21, a surrogate molecule was developed for use in preclinical disease models. The Ab728 antibody is a murine IgG1 monoclonal antibody that specifically binds to mouse IL-21. The binding affinity of murine IL-21 to Ab728 is 1 pM. Ab728 was able to completely neutralize murine IL-21 in in vivo and in vitro assays.

The non-obese diabetic (NOD) mouse is widely used as a model for Sjogren's Syndrome because it spontaneously develops lymphocytic infiltration in the salivary glands. Previous work showed that local suppression of IL-21 levels in the submandibular glands of NOD mice with shRNA lentivirus IL-21 could delay the development of symptoms similar to Sjogren's Syndrome (Liu H, et al. Local suppression of IL-21 in submandibular glands retard the development of Sjogren's syndrome in non-obese diabetic mice. J Oral Pathol Med 2012; 41: 728). The aim of this experiment was to investigate whether systemic administration of Ab728, a surrogate for Ab327, could prevent or attenuate the development of Sjogren's Syndrome in NOD mice.

Female NOD mice were treated with Ab728 or with the mIgG1 isotype control (20 mg / kg / week) starting at 7 weeks of age. The mice were sacrificed at 18 weeks of age and the salivary glands were harvested. A piece of salivary gland was fixed with 1.6% PFA and 20% sucrose at 4 ° C overnight, soaked in OCT and stored at -80 ° C until immunofluorescence analysis. Another piece was frozen in liquid nitrogen for mRNA studies.

In NOD mice, focal inflammation in the submandibular salivary glands and lacrimal glands develops from about 8 weeks of age onwards. The foci appear comparable in structure and cellular composition with infiltrates found in some human salivary glands, with the presence of T and B cells. To investigate whether anti-IL-21 treatment alleviated lymphocytic infiltration in the NOD salivary gland, a a stain for immunofluorescence. Briefly, 8 pm frozen sections of salivary glands were washed with PBS and then incubated for 1 hour at room temperature with purified primary antibodies, followed by incubation with the appropriate labeled secondary antibodies for 30 minutes. The primary antibodies were anti-CD3 (T cells) and anti-B220 (B cells) from BD Biosciences. Secondary antibodies were Alexa Fluor 488 goat anti-rat IgG and DyLight 594 goat anti-Armenian hamster from Jackson ImmunoResearch Laboratories. DAPI was used to identify the nucleus of the cells.

NOD mice treated with control mIgGI antibody showed the presence of typical lymphocytic infiltrates arranged as periductal aggregates with highly organized T and B lymphocytes (Figure 6) that resemble lymphocytic foci found in patients with Sjogren's syndrome. Treatment with Ab728 effectively decreased not only the number but also the size of the foci observed.

The development of lymphoid aggregates in Sjogren's syndrome is believed to be regulated by the ectopic production of the lymphoid chemokine CXCL13 and its cognate receptor CXCR5, which regulate the recirculation and positioning of B cells and CD4 + follicular T cells (TFH). in the structures of the germinal center. IL-21 has been shown to be involved in the maintenance of TFH and the structures of the germinal center. IL-21 also controls the activation of CD8 + T lymphocytes, which are believed to destroy target cells through perforin and granzymes. To investigate whether anti-IL-21 treatment would decrease the expression of these markers, total RNA was isolated from frozen salivary glands by homogenization in Trizol followed by the RNeasy Mini kit (Qiagen, Inc.). RNA concentrations were determined from spectrophotometric absorption at 260 nm. RNA was reverse transcribed into cDNA using the High Throughput cDNA Reverse Transcription Kit (PE Applied Biosystems). All reactions were performed in triplicate to determine the relative abundance of analyzed mRNA. The primer probe sets for IL-21 (Mm00517640 m1), CXCR5 (Mm00432086_m1), CXCL13 (Mm04214185_s1), CCR9 (Mm02620030_s1), Granzyme B (Mm00442834_m1), and CD8 (MmOl) 182107 were obtained from PE182107. GusB (Mm00446956_m1) was measured as endogenous controls to normalize variability in gene expression levels. Expression data was analyzed using the Delta Ct procedure. Individual Ct values were calculated as means of triplicate measurements.

CXCL13, CXCR5, IL-21, CD8, and Granzyme B mRNA transcripts were statistically significantly down-regulated in Ab728-treated mice, compared to control mIgG1 antibody-treated mice. Figure 5 shows the analysis of mRNA in salivary glands of mice. It can be seen that the treatment modulates the expression of proteins involved in the disease. In summary, administration of a mouse anti-IL-21 antibody (Ab728) decreased lymphocytic infiltration in the salivary glands and delayed the development of SS-like symptoms in NOD mice.

Anti-mIL-21 (Ab728) treatment prevents diabetes in NOD mice.

Ab728 is a murine IgG1 monoclonal antibody that specifically binds to mouse IL-21. The binding affinity of murine IL-21 to replace Ab728 is 1 pM. Ab728 was able to completely neutralize murine IL-21 in in vivo and in vitro assays.

Human type I diabetes is an autoimmune disease that results from the autoreactive destruction of insulin-producing beta cells in the islets of Langerhans of the pancreas, leading to the consequent loss of insulin production. The non-obese diabetic (NOD) mouse strain develops a similar disease and also serves as a model system to study the mechanisms involved in the initiation and propagation of the autoimmune response. Histological studies have shown that few immune cell infiltrates are seen in the islets until approximately 3 to 4 weeks of age, when male and female mice begin to demonstrate mononuclear infiltrates surrounding the islet (periinsulitis). These infiltrates progress and invade the islets (insulitis) followed by hyperglycemia and fully developed diabetes beginning at approximately 12 weeks of age.

Previous work showed that elimination of IL-21 signaling in NOD mice leads to almost complete abrogation of disease development (Spolski R, et al. IL-21 signaling is critical for the development of type I diabetes in the NOD mouse. Proc Natl Acad Sci USA 2008; 105: 14028, 2008). The purpose of the experiment was to investigate whether systemic administration of Ab728 was able to prevent or attenuate the development of diabetes in NOD mice.

Female NOD mice were treated with Ab728 or the mIgG1 isotype control (20 mg / kg / week) at different time periods in the disease process. One group of mice began treatment at 7 weeks of age (prevention study, Figure 7A) and another group of animals began treatment at 13 weeks of age (late preclinical stage, Figure 7B). In both situations, the mice were followed for the development of diabetes until the mice were 37 weeks old. To follow the development of diabetes, blood glucose levels were monitored weekly and animals were considered diabetic if the blood glucose was above 250 mg / dl in two consecutive measurements. Exposure to Ab728 was confirmed by quantitative ELISA.

NOD mice treated with the control antibody mIgG1 began to develop diabetes when they were 13-15 weeks old, and 75% of the mice progressed to overt diabetes at 37 weeks (9 out of 12 preventive mice and 6 out of 8 mice for the late preclinical stage - see Figure 7A and Figure 7B). In contrast, anti-IL-21 treatment significantly delayed the progression of diabetes. Only one in 12 mice (8%, Figure 7A, p = 0.0007) developed diabetes when treatment began at 7 weeks of age and only one in eleven mice (9%, Figure 7B, p = 0.002) progressed Overt diabetes when treatment began during the late preclinical stage.

In summary, administration of an anti-mouse IL-21 antibody (Ab728) effectively prevented the development of diabetes in NOD mice.

Sequences

Ab327 amino acid sequences

(continuation)

The following sequences were used in the Example and the Assays.

IL-21 HUMANA - UniprotKB / Swiss-Prot database entry # Q9HBE4 QDRHMIRMRQLIDIVDQLKNYVNDLVPEFLPAPEDVETN CE WS AFSCFQKAQLK SANTGNNERIINVSIKKLKRKPPSTNAGRKRKYEFLERPSCDLQLVPEFLPSCDSKPGPTHLERPSK 13SKEFLKRKPPSTNAGRKKHEDLERPSCDLPG

IL-21 CYNO - The sequence was internally cloned not available in the public database QDRFIMIRMRQLIDIYDQLKN Y YN DLD PEFLP APED VETN CE WS AI SCF QKAQLKS ANTGNNERIINLSIKKLKRKSPSTGAERRQKHRLSTCPSCDSYEKKFQHPKEFQLSGPSCDSYEKKFQHPKEFSG 14

IL-21 MOUSE - UniprotKB / Swiss-Prot database entry # Q9ES17 HKSSPQGPDRLLIRLRHLIDIVEQLKIYENDLDPELLSAPQDVKGHCEHAAFACFQ KAKLKPSNPGNNKTFIIDLVAQLRRWRLPARRKGKKPSQKHLIDIVEQLKIYENDLDPELLSAPQDVKGHCEHAAFACFQ KAKLKPSNPGNNKTFIIDLVAQLRRWRLPARRKGKKPSQKS FLQLRWRLPARRKGKPSQKHL 15

IL-21 RATA - UnirotKB / Swiss-Prot database entry # A3QPB9 HKSSPQRPDHLLIRLRHLMDIVEQLKIYENDLDPELLTAPQDVKGQCEHEAFACF QKAKLKPSNTGNNKTFINDLLAQLRRKLPSAKRTGNTP1 KQLCRWLPAKRTGNKQLCRWLPAKRTGNTP1)

IL-21 RABBIT - (COMMERCIAL REAGENT - R&D Systems, cat # 7274-RB / CF

EIKS S SKG QDRYMIRMEIQLLDIVDQLQ SD VNDLDPDFLP APQD V QKG CEQ S AF S C F QKAQLKP AN AGDN GKRIS SLIKQLKRKLPSTKSKKT QKHRPT CP S C Y S YEKKNL KEFLERLKSLQHIQKM1HLR: 17

DNA for Ab327 expression

Claims (14)

1. An antibody that binds to human IL-21 comprising a heavy chain variable region (HCVR) and a light chain variable region (LCVR), wherein the lCv R comprises SEQ ID NO: 7 in Cd Rl 1, Se Q ID n O: 8 in CDRL2 and SEQ ID NO: 9 in CDRL3 and wherein the HCVR comprises SEQ ID NO: 10 in CDRH1, SEQ ID NO: 11 in CDRH2 and SEQ ID NO: 12 in CDRh 3 , wherein the heavy chain comprises an HCVR having SEQ ID NO: 1 and wherein the light chain comprises an LCVR having SEQ.ID.NO: 2. 2. An antibody according to claim 1 consisting of two antibody heavy chains and two antibody light chains, in which each heavy chain comprises a heavy chain variable domain, the amino acid sequence of which is the sequence of SEQ ID NO : 1, and each light chain comprises a light chain variable domain, whose amino acid sequence is the sequence of SEQ ID NO: 2. The antibody according to claim 1 or claim 2, wherein the amino acid sequence of each heavy chain is the sequence of SEQ ID NO: 3 and the amino acid sequence of each light chain is the sequence of SEQ ID NO: 4. 4. A DNA molecule comprising a polynucleotide encoding the heavy chain of the antibody whose amino acid sequence is the sequence of SEQ ID NO: 3 and comprising a polynucleotide encoding the light chain of the antibody whose amino acid sequence is the sequence of SEQ ID NO: 4. The DNA molecule of claim 4, wherein the sequence of the polynucleotide encoding the heavy chain of the antibody is the sequence of SEQ ID NO: 5 and wherein the sequence of the polynucleotide encoding the light chain of the antibody is the sequence of SEQ ID NO: 6. 6. A mammalian cell transformed with the DNA molecule of claim 4 or claim 5, wherein the transformed mammalian cell is capable of expressing an antibody comprising two antibody heavy chains and two immunoglobulin light chains, wherein the The amino acid sequence of each of the two heavy chains is the sequence of SEQ ID NO: 3 and the amino acid sequence of each of the two light chains is the sequence of SEQ ID NO: 4. 7. A method of producing an antibody, the antibody of which comprises two antibody heavy chains and two immunoglobulin light chains, wherein the amino acid sequence of each of the two heavy chains is the sequence of SEQ ID NO: 3 and the amino acid sequence of each of the two light chains is the sequence of SEQ ID NO: 4, and which method comprises: to. culturing the mammalian cell of claim 6 under conditions such that the antibody is expressed, and b. recovering the expressed antibody. 8. An antibody obtainable by the method of claim 7. The antibody of any one of claims 1 to 3 or 8 for use in therapy. The antibody for use according to claim 9, wherein the therapy is the treatment of an autoimmune condition. The antibody for use according to claim 9, wherein the therapy is the treatment of primary Sjogren's syndrome, Sjogren's syndrome, systemic lupus erythematosus, Grave's disease or type 1 diabetes. The antibody for use according to claim 9, wherein the therapy is the treatment of primary Sjogren's Syndrome or Sjogren's Syndrome. The antibody for use according to claim 9, wherein the therapy is the treatment of systemic lupus erythematosus. 14. A pharmaceutical composition comprising the antibody of any one of claims 1 to 3 or 8 and a pharmaceutically acceptable excipient.